Fluorescent dyes are known to be particularly suitable for biological applications in which a highly sensitive detection method is desirable. By binding to a specific biological ingredient in a sample, a fluorescent dye can be used to indicate the presence or the quantity of the specific ingredient in a sample.
A variety of fluorescent dyes are commercially available for specific fluorescent staining and quantitation of DNA and RNA, and other applications involving nucleic acids, see e.g. Haugland, HANDBOOK OF FLUORESCENT PROBES AND RESEARCH CHEMICALS, Set 31 (5th Ed. 1992, Molecular Probes, Inc., Eugene, Oreg.) (incorporated by reference). Among these are derivatives of 5-substituted-3,8-diamino-6-phenylphenanthridium (5-DAPP): ##STR2## When R is ethyl, the dye is commonly called ethidium. When it is methyl, the dye is methidium. If R is 3-(N,N-diethyl-N-methylammonium)propyl, the dye is propidium. Various other analogs of 5-DAPP are known, including symmetric or asymmetric dimeric derivatives, derivatives in which the position(s) of the amino groups are changed or the amino groups are further modified by chemical substitution or omission and derivatives in which the aryl moiety is replaced by modified aryl, alkyl, arylalkyl or heteroaryl groups or by hydrogen.
Ethidium and its analogs were originally studied in the early 1940's as therapeutic agents for treatment of trypanosomiasis. They were subsequently found to bind to nucleic acids. This binding can be detected by the change in fluorescence properties of the nucleic acid complex following dye binding. When bound to nucleic acids, the 5-DAPP dyes generally have desirable fluorescence spectral properties. In particular, they absorb light at 488 nm and 514 nm, making them useful with instrumentation that uses the argon laser as an excitation source, such as flow cytometers and laser scanning microscopes. Furthermore, the fluorescence emission of the 5-DAPP dyes has a Stokes shift that is sufficient to permit detection of their fluorescence usually at a wavelength beyond about 580 nm, so that cellular autofluorescence is reduced. This characteristic also makes a 5-DAPP dye useful for multicolor applications in conjunction with a second dye, such as fluorescein or one of its conjugates, that is excited by the same source, but whose fluorescence is optimally detected at a shorter wavelength (typically at less than about 540 nm).
Not all nucleic acid stains can be used with living cells. To be useful for the analysis of nucleic acids in living cells, a detection reagent must be able to enter living cells and to respond to the presence of nucleic acids. It is particularly important to be able to stain nucleic acids in viable cells if it is desired to analyze and, if needed, sort viable cells according to the nucleic acid content or proliferative state of these cells based on their fluorescence. It is furthermore of importance to retain the cell viability if one wishes to sort and clone cells based on some additional fluorescence parameter.
It is generally recognized that ethidium and its analogs are usually not suitable for staining of nucleic acids in living cells in which the cell membrane is intact, except for permeabilized cells or at very high dye concentrations. Consequently several of these probes, in particular ethidium bromide (Tanke, et al., J. IMMUNOL. METH. 52, 91 (1982)), propidium iodide (U.S. Pat. No. 5,057,413 to Terstappen et al. Oct. 15, 1991 U.S. Pat. No. 5,314,805) and ethidium homodimer (Live/Dead.RTM. kit, U.S. Ser. No. 07/783,182 to Haugland, et al., filed Oct. 26, 1991) U.S. Pat. No. 5,314,805, have been used extensively to detect and quantitate cells in which the membrane is compromised or missing, i.e. dead cells.
Making the 5-DAPP dyes more useful for staining nucleic acids in a wide variety of living cells requires improving access of the dyes to intracellular nucleic acids. Although numerous methods for enhancing permeability of organic compounds into cells have been described, including chemical- or electro-permeabilization, scrape loading, use of detergents, microinjection or various means of mechanical disruption, all of these methods intrinsically have the potential disadvantage of altering the properties of the cell membrane and thus the cell's intrinsic properties or proliferative capacity. Furthermore it is often difficult to achieve uniform labeling or reproducibility using these methods and some of the methods such as microinjection are not technically feasible on very small or fragile cells.
One method for improving the uptake of ethidium into living cells involves the chemical reduction of ethidium bromide to a dihydrophenanthridine derivative with no positive charge, see e.g. Bucana, et al., J. HISTOCHEM. & CYTOCHEM. 34, 1109 (1986). Unlike ethidium or the subject materials, this compound does not bind to nucleic acids and requires the secondary step of intracellular oxidation to regenerate ethidium intracellularly. Although the ultimate result in certain types of living cells is nuclear staining by a 5-DAPP derivative, not all cells are capable of oxidizing this type of dihydrophenanthridine to the nucleic acid stain.
This invention describes an effective means for improving uptake and staining of certain 3,8-diaminophenanthridium dyes in a wide variety of viable cells in culture or tissues by slightly increasing the size of the quaternizing substituent at the 5-position of the phenanthridine ring and thereby increasing the lipophilicity of the probe. Although only a slight chemical modification, this change significantly improves the permeability of the dye through the membrane of living cells and thus the staining of viable cells without significantly altering the ability of the dye to stain nucleic acids and without appreciably altering the spectral properties of the complex. There is an optimal size of the quaternizing substituent in that a further increase in size of the substituent has a deleterious effect on the nucleic acid staining by the dye. These reagents enable the staining of nucleic acids in living cells by a simple incubation with the reagent in standard culture medium, or in vivo by injection in a suitable biologically compatible fluid such as saline without resorting to use of harsh additives. Furthermore, use of these reagents permits detection, analysis and, if required, sorting of the viable cells based on the fluorescence intensity of their complex with nucleic acids, or based on fluorescence polarization, excited state fluorescence lifetime, or other dye-nucleic acid complex-related optical properties.
The subject dyes have the same basic 5-DAPP structure as does ethidium, except that instead of the two-carbon alkyl group as in ethidium, the substituent R at the 5-position of the phenanthridium ring contains 4 or more carbon atoms. Synthesis of a few examples of 5-DAPP molecules for use as drugs has been described in earlier publications, e.g. Watkins, J. CHEM SOC. 3059 (1952) (incorporated by reference). This paper and a related paper, Watkins & Woolfe, NATURE 169, 506 (1952) compare the use of various derivatives of 5-DAPP as trypanocides and conclude that the therapeutic potential of ethidium is superior to that of other 5-DAPP derivatives with longer alkyl chains. The Watkins paper speculates that the concentration of the free-base form of ethidium at pH 6-9 is greater than that of methidium (which is shown to be less effective than ethidium) and that this property may result in an increased rate of diffusion of the ethidium versus methidium across the cell membrane into the trypanosome cytoplasm. Watkins does not, however, indicate that other less effective 5-DAPP derivatives would diffuse across the cell membrane more quickly than ethidium or that they would be useful as fluorescent detection reagents for nucleic acids in living cells.
A application Ser. No. 08/047,683, abandoned (incorporated by reference) describes the use of the subject dyes to distinguish between live gram-positive and live gram-negative bacteria. Although dyes such as hexidium also lightly stain some gram negative organisms when used alone, the staining of gram-positive cells is significantly greater. This discrimination is not possible with ethidium, which does not effectively stain either live gram negative or live gram positive bacteria under the same mild conditions.